This proposal describes our continuing efforts to determine the role played by pteridine and folate coenzymes or substrates in enzymatic systems. Structural factors commonly present in such naturally occurring pteridines include 2-amino and 4-hydroxy substituents, a reduced pyrazine ring and an alkyl substituent at the 5-position. During the course of a pteridine -mediated biological reaction, oxidation often occurs and in some cases there is also a loss of the 5-substituent. To investigate the biochemistry of these compounds the program has been divided into three major sections: (a) The efficient, quantitative isolation of pteridine requiring enzymes, such as the dihydrofolate and dihydropteridine reductases, and thymidylate synthetase, in bulk amounts for structural characterization purposes, using existing and new affinity chromatographic procedures; (b) The use of fluorescent,photoactive, and mercurated substrate or cofactor analogs (e.g., fluorescein derivatives of Methotrexate (MTX) and 5-fluoro-2' -deoxyuridylate (FdUMP), or 5'azidophenol FdUMP) of these enzymes, as structural probes; and (c) A study of the chemistry of the reduced and substituted pteridines using H1 and C13 nuclear magnetic resonance, and rapid reaction techniques (e.g., stop-flow spectrophotometry), with particular emphasis on the structural changes occurring when oxygen is incorporated, and on factors which induce lability into the 5-substituents. Also described is the preparation and characterization of two new types of folate antagonists. In one case MTX is bound to a series of high-molecular weight carriers such as soluble starch, dextrans, murine, and bovine serum albumin, in the second case MTX is masked by acylation and amide formation. Their effectiveness as therapeutic cytological agents is being determined against the L1210 ascites tumor in BDF1 mice, and against MTX-resistant and non-resistant in vitro cell cultures from the same source.